Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells.

BACKGROUND: A major problem in the healing of bone defects is insufficient or absent blood supply within the defect. To overcome this challenging problem, a plethora of approaches within bone tissue engineering have been developed recently. Bearing in mind that the interplay of various diffusible factors released by endothelial cells (ECs) and osteoblasts (OBs) have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions, we set the focus on the simultaneous application of these cell types together with platelet-rich plasma (PRP) as a growth factor reservoir within ectopic bone tissue engineering constructs. AIM: To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells (ASCs) induced into ECs and OBs. METHODS: ASCs isolated from adipose tissue, induced in vitro into ECs, OBs or just expanded were used for implant construction as followed: BPEO, endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix; BPUI, uninduced ASCs with PRP and bone mineral matrix; BC (control), only bone mineral matrix. At 1, 2, 4 and 8 wk after subcutaneous implantation in mice, implants were extracted and endothelial-related and bone-related gene expression were analyzed, while histological analyses were performed after 2 and 8 wk. RESULTS: The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation. BC had the lowest endothelial-related gene expression, weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO. Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO. BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs. Except Vwf, endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk. Tissue regression was noticed in BPEO constructs after 8 wk. CONCLUSION: Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis, but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.

Macrophages' contribution to ectopic osteogenesis in combination with blood clot and bone substitute: possibility for application in bone regeneration strategies.

PURPOSE: Given the great potential of macrophages in the processes of tissue repair and regeneration, the aim of our study was to examine the contribution that macrophages will have in osteogenic process when combined and implanted with blood clot (BC) and mineral bone substitute (MBS) in mice subcutaneous implantation model. METHODS: Three types of implants were constructed and implanted subcutaneously into BALB/c mice: (1) RMBM implants (made of resident tissue macrophages, BC and MBS), (2) BM implants (made of BC and MBS), and (3) M implants (made of MBS only) where the last two served as control implants. One, two, four and eight weeks after implantation implants were explanted, and histochemical, immunohistochemical, and histomorphometric analyses were performed. RESULTS: Increased vascularization, particularly pronounced two and four weeks after implantation and pronounced tissue infiltration in eight week term in RMBM implants compared with both other types, likewise the presence of osteoblast-like cells, osteoid-like structures, and more prominent osteopontin and osteocalcin immunoexpression in RMBM implants indicated more pronounced osteogenic process within them. CONCLUSION: Our results suggest that macrophages deserve to be considered as a cell component when constructing implants in bone regenerative medicine strategies to improve bone fracture healing process.

Osteogenic capacity of diluted platelet-rich plasma in ectopic bone-forming model: Benefits for bone regeneration.

Platelet-rich plasma (PRP) with normal and below-normal physiological concentrations of platelets is designated as diluted PRP (dPRP). The aims of this study are to evaluate whether bone mineral matrix in combination with dPRP possesses osteogenic capacity; and whether the differences in dynamics and osteogenic process pattern depend on different platelet concentrations, to what extent, and also what could be benefits for bone regeneration in clinical practice. Three types of implants were made: BMM-bone mineral matrix alone; dPRP/10-bone mineral matrix mixed with dPRP (concentration of platelets 10 times lower than physiological level) and dPRP/3-bone mineral matrix mixed with dPRP (concentration of platelets 3 times lower than physiological level). A subcutaneous implantation model in Balb/c mice was used. The implants were analyzed using expression analysis of bone-related genes, histochemical, immunohistochemical and histomorphometrical analysis. All types of implants induced creation of necessary preconditions for supporting osteogenic processes, but did not induce visible young bone growth. Implant types dPRP/10 and dPRP/3 showed very similar and significantly better stimulatory effects on osteogenic processes than bone matrix alone. In this study, significant ectopic osteogenic potential of concentration of platelets in PRP that are lower than physiological level in blood plasma in combination with bone mineral matrix was demonstrated. Diluted platelet-rich plasma could be a promising and useful adjuvant therapeutic agent in bone regeneration.

Ectopic osteogenic capacity of freshly isolated adipose-derived stromal vascular fraction cells supported with platelet-rich plasma: A simulation of intraoperative procedure.

Bone defects represent a serious problem in cranio-maxillofacial surgery. Autologous adipose-derived stromal vascular fraction (SVF) cells in combination with biological factors and bone substitutes were previously proposed as alternative to bone grafting. By simulating an intraoperative procedure we examined osteogenic capacity of the combination of two autologous components, freshly isolated adipose-derived SVF cells, and platelet-rich plasma (PRP), delivered on bone mineral matrix (BMM) carrier (SPB group) in mice ectopic bone forming model. Implantation of BMM only (B group) was a control. The presence of adipose-derived stem cells (ADSCs) in SVF was detected by immunocytochemical analysis. Expression of bone- and endothelial-related genes was compared between freshly isolated SVF and ADSCs obtained from SVF after in vitro cultivation. The implants were analyzed using expression analysis of bone-related genes at one, two, four and eight weeks and histochemical, immunohistochemical and histomorphometrical analyses at two and eight weeks after implantation. Freshly isolated adipose-derived SVF contained ADSCs and exhibited promising osteogenic and vasculogenic capacity. At two and four weeks, significantly higher expression of bone-related genes was detected in SPB group compared to B group. The signs of osteogenic process were more pronounced in SPB than in B implants. By the end of experiment, percentage of infiltrated tissue and vascularization was significantly higher in SPB than in B implants. Adipose-derived SVF cells, PRP and BMM rapidly initiated osteogenesis what makes this combination promising candidate for treatment of bone defects.

Osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells combined with platelet-rich plasma in an ectopic model.

PURPOSE: The osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells (ADSCs) combined with platelet-rich plasma (PRP) and implanted on bone mineral matrix (BMM) carrier was examined in a subcutaneous model in Balb/c mice. METHODS: In vitro osteogenic differentiation of ADSCs was monitored by relative bone-related gene expression and osteocalcin expression at the third, seventh and 15th day. Test implants consisting of in vitro osteo-induced ADSCs, PRP and BMM (OPC implants) and control implants consisting of PRP and BMM (PC implants) were examined. The relative expression of the bone-related genes encoding osterix, osteocalcin, collagen type I α1 and alkaline phosphatase was examined in implants extracted at one, two, four and eight weeks. Histochemical, immunohistochemical and histomorphometric analyses of implants extracted at two and eight weeks were performed. RESULTS: The highest relative expression of bone-related genes and osteocalcin expression was found at the 15th day of in vitro osteogenic induction of the ADSCs. Permanent and continuous increased expression of bone-related genes was noticed in OPC implants at eight weeks. Expression peaks of bone-related genes in PC implants were at two and four weeks, but they significantly decreased at eight weeks. The signs of resorption, formation of callus-like tissue positive for osteocalcin and increased presence of bone cells were found in OPC implants compared with PC implants. A higher percentage of infiltrated tissue and vascularisation was found in OPC than in PC implants. CONCLUSIONS: The combination of in vitro osteo-induced ADSCs and PRP on BMM carrier represents a promising approach for bone regeneration.